This invention relates generally to the field of immunological reactions in biological fluids and, more particularly, to a method and comparison useful in preparing red blood cells for use as indicator cells to detect the presence of antigens and antibodies in biological fluids.
The traditional method of determining ABO and Rh grouping, antibody screening, antibody identification and cross-matching is biological fluids such as blood, serum, and saliva, is through agglutination reactions. Until relatively recently, agglutination tests have been limited by their need to be performed manually. One of the primary reasons for requiring manual observation is the lack of an easily discernible end point. Various reagents are now available to increase the sensitivity and reliability of agglutination reactions to permit at least semi-automation. Even with the improved techniques, agglutination reactions still face the drawback of lacking an objective end point. Sophisticated electronic equipment partially compensates for this drawback, but such equipment is expensive and is not feasible for many smaller laboratories.
It has long been known that antibodies and antigens may be absorbed onto the surface of erythrocytes to detect the presence of immunoglobulins and anti-immunoglobulins in biological fluids utilizing passive agglutination reactions. The protein antigens and antibodies will not immunologically adhere to the erythrocytes unless the cell structure is altered or coupling agents are employed. Once suitable modification of the red cells is achieved and antigens of antibodies are then attached, it it known to utilize the resulting indicator red blood cells to determine the presence of antigens and antibodies in biological fluids using passive agglutination reactions. These reactions have most frequently been carried out in liquid phase assays. Even when improved indicator red blood cells of the type described above are employed in liquid phase assays, some of the same problems associated with conventional agglutination reactions are encountered including lack of sensitivity and difficulty in determining end points. A technique for utilizing red blood cells as indicators of an immunological reaction in liquid phase assays is discussed in detail in the book "Immuno Assays for the 80s" by A. Voller et al., especially Chapter 3 by R. R. A. Coombs, published by University Park Press, Baltimore, Maryland, 1981. Even utilizing the improved techniques of Coombs and other pioneers in the field, employment of indicator red blood cells in liquid phase serology is still limited by the lack of a clearly discernible and objective end point.
A considerable advance in the art of blood group serology is represented by U.S. Pat. No. 4,275,053 to Rosenfield issued June 23, 1981. This patent is directed to a procedure for conducting blood group serology in solid phase. The disadvantage of liquid phase hemagglutination tests as well as the advantages of working in the solid phase are discussed in detail in the referenced patent. The single greatest advantage of solid phase assays is the use of immune adherence rather than hemagglutination for the detection of antigen-antibody reactions. This is a much more objective and discernible end point for either manual or automated reading.
A further advantage in solid phase assays for determining the presence of antibodies and antigens in biological fluids is described in U.S. Pat. No. 4,608,246 to Bayer, Plapp, Sinor et al., issued Aug. 26, 1986. In the Bayer, Plapp, Sinor et al. patent, a technique for broad application of solid phase blood group serology is described. The method disclosed offers numerous advantages over the original concept described in the Rosenfield patent including applicability to a broader range of substrates, fewer steps, longer shelf life, and a shorter time for carrying out the tests. A distinguishing characteristic of the Bayer, Plapp, Sinor et al. method is the utilization of proteolytic enzymes to activate the red blood cells prior to subjecting them to an immunological reaction.
When utilizing any of the prior art techniques for modifying red cell structures in order to bind antibodies and utilize the cells as indicator cells in liquid phase technology, the agent utilized to modify the cells tends to render the cells "sticky". When these indicator red blood cells are used in solid phase red cell adherence assays, this "stickiness" prevents a clear cut distinction between positive and negative results. Even utilizing enzyme activation as taught by Bayer, Plapp, Sinor, et al., preparation of the indicator cells is time consuming, requires large amounts of antibody, and the shelf life of indicator cells is relatively short. Also, the enzyme treatment as well as other activation techniques may destroy some antigens.